Cultured tobacco cells as a matrix for consumable products

ABSTRACT

Various embodiments are directed to consumable products incorporating a matrix of cultured tobacco cells. Any tobacco variety may be utilized for establishing in vitro tobacco cultures, including native tobacco varieties and genetically modified tobacco varieties derived from any tobacco variety. Various embodiments are directed to methods for producing tobacco products incorporating cellular material and/or extracts derived from tobacco cells cultured in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/318,209, filed on Mar. 26, 2010, the contents of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND

Conventionally-grown tobacco tends to be dark in color which is notalways desirable. Thus, a need exists for a consumable product lackingdark pigments

SUMMARY

One embodiment of the invention is a method for producing a product,comprising the steps of growing tobacco cells in liquid suspensionculture under conditpions of osmotic stress and/or saline stress to forma culture material comprising tobacco cells and extracellular matrix;harvesting culture material; drying the culture material to create atobacco matrix; and incorporating the tobacco matrix into a product.

Another embodiment of the invention is consumable product comprising amatrix of cultured tobacco cells, wherein the matrix of cultured tobaccocells comprises at least 5% by weight extracellular matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an image of exemplary freeze-dried, tobacco cellscultured in vitro that can be incorporated into various smokelessproducts, as a substitute for conventional tobacco particles, shreds,fine cut or long cut tobacco lamina, and/or as an additive ingredient.

FIG. 2 shows an exemplary method for establishing in vitro cultures oftobacco cells.

FIG. 3 shows an exemplary method for preparing tobacco matrix materialfrom cultured tobacco cells established according to the methodillustrated in FIG. 2.

DETAILED DESCRIPTION

Some factors essential for controlling cellular growth anddifferentiation have been defined, including various plant growthhormones and differentiation factors that operate alone or insynergistic combinations. For example, by controlling the relative ratioof cytokinin to auxin concentration in a culture medium, in vitrocultures of plant cell suspension exhibiting various properties ofinterest can be generated. Typically, a callus culture can beestablished from an initial tissue explant removed from a plant ofinterest. Culture conditions suitable for initiating and maintaining theproliferative state of callus cultures can be defined experimentally forany plant of interest, such as tobacco plants.

Various embodiments are directed to lit-end (i.e., smokeable) andsmokeless products incorporating culture material (including cellularmaterial and/or secreted extracellular material) from tobacco cellscultured in vitro. Any tobacco variety may be utilized for establishingin vitro tobacco cultures, including native tobacco varieties andgenetically modified tobacco varieties derived from any tobacco variety,and can be maintained for long periods under proper conditions. Variousembodiments are directed to methods for producing smokeless productsincorporating culture material from tobacco cells cultured in vitro.Tobacco-based products incorporating various alternative ingredientsthat can improve the enjoyment of the products, and that can be producedin a cost-effective manner, are highly desirable.

A. Definitions

The term “non-tobacco smoking material” or “tobacco substitute” refersto a material other than tobacco utilized for making smoking articlesand smokeless products. For example, the common non-tobacco fillers areorganic plant constituents comprising cellulose, includingvegetable-based substances, such as rhubarb, plantain, coltsfoot,self-heal, comfrey, stinging nettle, watercress, groundsel, oxtongue,beet, mallow, poppy leaves, sage, walnut, cherry, red beech, hornbeam,maple, hazlenut, goldenregen, prickly broom, eucalyptus, bagasse,lettuce, peanut, soybean, potato, corn, yam, taro, and cocoa, paper, andseaweed.

The term “tobacco extender” or “tobacco supplement” refers to anontobacco material, natural or synthetic, that can be blended withtobacco in making of smoking articles and smokeless products to producea desired effect, such as cost savings, alteration of smokeconstituents, reduction of tar delivery, improvement of physicalcharacteristics of the products.

The term “tobacco dust” refers to ground tobacco and/or tobaccoparticles created by tobacco breakage during manufacturing processes.

The term “reconstituted tobacco” refers to tobacco dust, stems,by-products, and equivalents, that may be mixed with a cohesive agent,and that are rolled or cast into a flat sheet of uniform thickness andquality. A sheet of reconstituted tobacco may be cut into any sizeshreds. Examples of basic sheet processes include dust-impingementprocess, tobacco-slurry process, impregnation-of-web process, paperprocess, and extrusion process.

The term “hydroscopic agent” or “plasticizer” refers to humectants andequivalent agents added to tobacco or substituted tobacco to help retainmoisture and plasticity, such as glycerin.

The term “without added light” means substantially without exposure tosunlight and with only incidental exposure to artificial light.Preferably, when a plant cell culture is grown without added light, acarbon source (sucrose, for example) is provided in the culture medium.

As used herein, the term “extracellular matrix” denotes materialsecreted by plant cells. Exemplary components of extracellular matrixinclude carbohydrates (such as polysaccharides), proteins, andglycoproteins such as proteoglycans. More particularly, exemplarycomponents of extracellular matrix include cellulose, hemicellulose,pectin, and waxes. Additional exemplary components of extracellularmatrix are disclosed in U.S. Patent Application Publication No.2009/0038033, incorporated herein by reference.

As used herein, the term “osmotic stress” refers to culture conditionswherein the osmolarity is sufficiently higher than that needed for themaximum rate of increase in numbers of cells so as to result in ameasurable different in one or more cellular traits (such as rate ofgrowth) and/or the extracellular matrix.

As used herein, the term “saline stress” refers to culture conditionswherein the concentration of salt (including but not limited to NaCl) issufficiently higher than that needed for the maximum rate of increase innumbers of cells so as to result in a measurable different in one ormore cellular traits (such as rate of growth) and/or the extracellularmatrix.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

B. Methods for Establishing Cultures from Tobacco Explants

FIG. 1 illustrates an image of exemplary freeze-dried, tobacco cellscultured in vitro (“tobacco matrix material”) that can be incorporatedinto a consumable tobacco product, as a substitute for conventionaltobacco particles, shreds, fine cut or long cut tobacco lamina, and/oras an additive ingredient. In FIG. 1, the tobacco cells harvested fromin vitro cultures were subsequently freeze-dried to produce a lightweight, nearly white, and powdery substance that can be utilized forproducing various products of interest. Because the cultured tobaccocells are grown in the dark, the cultured tobacco cells appear nearlywhite, without the formation of dark pigments observed in traditionallygrown tobacco leaves. The disclosed tobacco matrix comprises culturedtobacco cells preferably along with extracellular matrix material. Thetobacco matrix, which includes tobacco cells as well as extracellularmatrix, is preferably substantially without dark pigmentation can beincorporated into various products of interest. A broad range ofproducts, exhibiting a number of attributes of interest, can be producedby varying certain parameters, including selecting suitable tobaccoexplants utilized to generate tobacco suspension cultures, growing thetobacco suspension cultures under suitable growth conditions, processingthe harvested material including tobacco cells by a suitable method, andincorporating the processed tobacco matrix material into various tobaccoproducts.

FIG. 2 shows an exemplary method for establishing in vitro cultures oftobacco cells that can be utilized for the preparation of a tobaccomatrix material. A subset of these steps may be sufficient to accomplishthe goals described herein. The exemplary method of FIG. 2 comprises:collecting seeds from a tobacco plant of interest and sterilizing theirexterior to eliminate unwanted organisms 200; planting seeds to grow atobacco plant of interest 210; removing tissue from the tobacco plant(for example, from a tobacco stem) for use as an explant 220;establishing a callus culture from the tobacco explant 230; establishinga cell suspension culture from the callus culture 240; and harvestingculture material (which includes tobacco cells and extracellular matrixmaterial) for further downstream processing steps 250.

Accordingly, in harvesting the cells, extracellular matrix associatedwith and/or secreted by the cells is preferably harvested with the cellsto aid in formation of the tobacco matrix material. Preferably theculture material, as measured after drying, includes about 5 to 80%percent secreted extracellular matrix, more preferably about 5 to 50%secreted extracellular matrix, still more preferably about 10 to 25%secreted extracellular matrix. The remainder of the culture material ispreferably mostly tobacco cells.

FIG. 3 shows an exemplary method for preparing tobacco matrix materialfrom cultured tobacco cells established according to the methodillustrated in FIG. 2. In FIG. 3, harvesting material from suspensioncultures 300; dehydrating harvested material to produce a tobacco matrixmaterial 310; and incorporating the dehydrated tobacco matrix materialinto a consumable tobacco product of interest 320, for example acigarette or pouched tobacco product.

For illustration, Examples 1-3, provided below, describe the compositionof an exemplary growth medium, growth conditions for establishingtobacco callus cultures, growth conditions for generating andsubculturing tobacco suspension cultures, conditions for harvesting oftobacco suspension cultures, and conditions for preparing a tobaccomatrix material.

Selection of Suitable Culture Media for Growing Tobacco Cells

Under controlled conditions, many types of in vitro cultures derivedfrom an explant derived from a tobacco plant of interest (“tobaccocultures”) can be stably established and indefinitely propagated.Tobacco cultures can grow in many types of media, including Murashigeand Skoog (MS), which is a commercially available, basic media commonlyutilized for supporting the growth of various plant-tissue cultures.Example 1, provided below, describes the preparation of the MS mediumand an exemplary method for preparing an explant from the stem of atobacco plant of interest. A callus culture can be established from theinitial explant, which can be propagated by subsequent rounds ofsubculturing as described in Example 1. Once established, the culturedtobacco cells exhibit characteristics of immortalized cells, and can bepropagated indefinitely in a culture medium, in which the cellulargrowth and division of the cultured cells are limited only by thereplenishment of fresh media.

In general, the explant is first introduced into an initiation media ina semi-solid gel form to support the development of solid cell masses(“callus”). A liquid media can be employed at later stages for growingnon-aggregated tobacco cell suspensions following the inoculation withthe pre-established tobacco callus. In general, the semi-solid gel mediacan be prepared by combining the components of the liquid media and agelling agent, such as agar, gellan gum, agarose, and GELRITE®. Methodsfor the preparation of such media are known to persons skilled in theart, and general formulations are described in U.S. Pat. Nos. 5,236,841;5,413,930; and 5,506,136, each is which is incorporated herein byreference. As further explained in Example 2, viable tobacco cellsuspensions cultures can be established by transferring a callus oftobacco cells into a liquid media, and can be grown in the dark withmoderate agitation.

Any steps equivalent to the described steps of the method describedherein are also contemplated, including individual steps and combinationof steps, known to persons skilled in the art. As one example, in orderto add flavors to the tobacco matrix, the culture may be grown in thepresence of a flavorant or a flavorant precursor which is taken up bythe cells and thence converted to a flavorant. As another example, acallus culture established from a tobacco explant, such as 230, and acell suspension culture established from a callus culture, such as 240,can be scaled-up in volume and number to accommodate manufacturinglevels of biomass (See FIG. 2). Cells can be cultivated in any volume,including small-scale cell cultures having a volume of less than about20 L. For example, a suitable volume includes an amount less than about1 L (e.g., 25 ml to 100 ml), from about 1 L to about 5 L, from about 5 Lto about 10 L, or from about 10 L to about 20 L. Alternatively, cellscan be cultivated in medium-scale cell cultures having a volume fromabout 20 L and about 500 L, from about 100 L to about 500 L, or fromabout 20 L to about 2,000 L. Alternatively, cells can be cultivated inlarge-scale bioreactors having a volume up to about 20,000 L, such asfrom about 1,000 L to about 10,000 L, from about 2,000 L to about 10,000L, or from about 1,000 L to about 20,000 L. In general, small-scaledevices are limited to a few liters in volume because they rely onsurface oxygen transfer to provide aeration for the cells. Examples ofsmall-scale devices include spinner flasks, T-flasks, and rollerbottles. Methods for establishing large-scale bioreactors such asstirred tank bioreactors are known by persons skilled in the art (seeArmstrong, U.S. Pat. No. 4,906,577 and Morrison, U.S. Pat. No.5,002,890, each of which is incorporated herein by reference).

Furthermore, the growth/maintenance of cultures and the harvesting oftobacco cells for further downstream processing steps, such as 250, canbe automated in part or in its entirety (See FIG. 2). As one method forgrowing the cell cultures in the dark, the cell suspensions can be grownin light-blocking containers to preclude the activation ofphoto-sensitive biochemical pathways that may lead to the production ofcolored pigments. Any light-blocking container can be utilized toreflect, scatter, and/or absorb light, and thus reduce and/or preventlight penetration into the container. Light-blocking containers can bemade by incorporating light blocking materials, as one or more coatingsor as laminates. A light-blocking material may include any suitablecolor dye. The light-blocking material can reflect or scatter the light,and/or can absorb the light to prevent exposure of the biological fluidinside the container to the light. The light-blocking material should becapable of blocking ultraviolet (UV) and/or visible light. For example,the light-blocking material can block light wavelengths from about 10 nmto about 1 mm, from about 10 nm to about 400 nm, or from about 400 nm toabout 750 nm, or from about 750 nm to about 1 mm. Light-blockingmaterials suitable for making cell culture containers are known topersons skilled in the art, including polymers, such as variouspolyesters. For example, polyethylene terephthalate (PET), polyethylenenaphthalate (PEN), polyvinyl fluoride (PVF), and tedlar PVF are highlyeffective in blocking ultraviolet (UV) light. Metals such as stainlesssteel, for example in a stainless steel bioreactor, are also effectivein blocking light.

Culture Conditions to Induce Changes in Cell Surface ExtracellularPolysaccharides and/or Polypeptides

Preferably, the culture medium is adapted to induce osmotic and salinestress to tobacco cells in suspension cultures in order to cause changesin the composition of the cell surface of cultured cells that promotecellular adhesion and/or the secretion of extracellular matrix.Increasing the amount of extracellular matrix present can aid in thesubsequent formation of a cohesive tobacco matrix. For example, theculture medium can be supplemented with an osmotic stress inducer, suchas polyethylene glycol (“PEG”), or a saline stress inducer, such asNaCl. For example, the culture medium in which the suspension culture isgrown can be supplemented with a sufficient amount of PEG or anequivalent, such as at least 10%, 15%, 20%, 25%, or 30%.

Alternatively, the culture medium can be supplemented with a sufficientamount of NaCl or an equivalent, selected from a range from about 300 mMto about 500 mM. Polyethylene glycol (PEG) can be also referred to aspolyethylene oxide (PEO) or polyoxyethylene (POE). The terms “PEG,”“PEO,” or “POE” may refer polymers of ethylene oxide. These terms can beinterchanged, however traditionally, PEG refers to polymers exhibiting amolecular mass below 20,000 g/mol; PEO refers to polymers exhibiting amolecular mass above 20,000 g/mol; and POE refers to a polymer of anymolecular mass.

Prior studies have shown that saline-adapted cells and PEG-adapted cellstransferred to iso-osmolar mannitol can release significantly morecarbohydrates and proteins from their cell walls into the culture mediumwhen compared to unadapted cells (See Iraki et al., “ExtracellularPolysaccharides and Proteins of Tobacco Cell Cultures and Changes inComposition Associated with Growth-limiting Adaptation to Water andSaline Stress,” Plant Physiology, Vol. 91: p 54-61 (1989), incorporatedherein by reference). There, tobacco cells (Nicotiana tabacum L. cv W38)were maintained in liquid suspension culture, such as MS medium andadapted to grow in a medium supplemented with either 30% PEG (−28 bar)or 428 mM NaC1 (−23 bar).

Selection of Suitable Explants from Tissues of Tobacco Plants

To provide a source of culture material (including cellular materialand/or secreted extracellular material), an in vitro culture of tobaccocells can be established from an “explant.” An explant is an organ,tissue, or cells derived from a plant, and are cultured in vitro for thepurpose of starting a plant-tissue culture. Suitable explants toinitiate in vitro cultures include any portion of a tobacco plant thatdemonstrates substantial metabolic activity, responsiveness to growthfactors, and accessibility by physical dissection. For establishingcultures for micro-propagation, the tissues most frequently utilized assuitable explants include meristematic ends of plants, such as the stemtip, auxiliary bud tip, and root tip. The meristematic ends can exhibithigh rates of cell division, and can either produce or store growthfactors necessary for growth and/or differentiation, including auxinsand cytokinins. In general, explants derived from aerial portions of aplant located above the soil may be preferable over root explantsbecause potentially contaminating microflora from aerial explants can beless difficult to remove than from root explants that can adhere moretightly to undesirable microflora. Alternatively, uncontaminatedexplants can be obtained by removing portions from seedlings that aregrown from surface-sterilized seeds. The hard surface of a seed is lesspermeable to penetration by more stringent sterilizing agents than moredelicate vegetative tissue.

Selection of Suitable Tobacco Varieties

In some embodiments, suitable tobacco explants can be derived from anynative tobacco variety selected from the genus Nicotiana, variousspecies of Nicotiana, including N. rustica and N. tabacum (e.g., LA B21,LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).Other species include N. acaulis, N. acuminata, N. acuminata var.multiflora, N. africana, N. alata, N. amplexicaulis, N. arentsii, N.attenuata, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis,N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N.excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N.goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N.knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N.megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia,N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N.paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N.quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp.ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N.spegazzinii, N. stocktonii, N. suaveolens, N. sylvestris, N.thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N.umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae.

In some embodiments, suitable tobacco explants can be derived fromnon-native tobacco plants, including genetically-modified tobacco plants(e.g., transgenic plants) derived from any tobacco variety, includingthe varieties provided above.

Suitable plants for transformation include any plant tissue capable oftransformation by various methods of transforming plants known bypersons skilled in the art, including electroporation, micro-projectilebombardment, and Agrobacterium-mediated transfer as described, forexample, in U.S. Pat. No. 4,459,355 that discloses a method fortransforming susceptible plants, including dicots, with an Agrobacteriumstrain containing a Ti plasmid; U.S. Pat. No. 4,795,855 that disclosestransformation of woody plants with an Agrobacterium vector; U.S. Pat.No. 4,940,838 that discloses a binary Agrobacterium vector; U.S. Pat.No. 4,945,050; and U.S. Pat. No. 5,015,580.

The herein-described tobacco matrix from cultured tobacco cells grown invitro provides various advantages. The tobacco matrix, particularly whengrown without added light, is substantially devoid of dark pigment thatoccurs in conventionally-grown tobacco plants, especially tobacco thathas been harvested and cured. This makes the cultured tobacco especiallydesirable for smokeless tobacco products, where the lack of pigment canbe easily observed.

Additionally, the cultured tobacco material exhibits a neutral flavorprofile, with little or none of the harsh, astringent, and/or bitterflavors normally found in conventionally-grown tobacco. This feature isespecially advantageous in view of the possibility that cultureconditions may be manipulated to achieve tobacco products that mayenhance the sensorial experience (e.g., flavor) of utilizing theproducts. The neutral flavor profile may make added flavors (whetheradded in culture or at a later stage) more readily detectable byconsumers.

Furthermore, a tobacco matrix as described herein may have a reducedlevel of undesired biological activity as compared toconventionally-grown tobacco: for example, reduced cytotoxicity and/orreduced mutagenicity. Cultured tobacco cells grown in a controlledenvironment are expected to have reduced levels of tobacco-specificnitrosamines (TSNAs) and reduced levels of heavy metals such as cadmium.

The cells grown in culture are capable of producing a range ofmetabolites observed in traditionally grown leaves (e.g., flavors,enzymes, amino acids, etc.), and can exhibit additional propertiesuseful for the improvement of tobacco products and/or development ofalternative tobacco products.

B. Products Incorporating Cultured Tobacco Material

Various embodiments are directed to consumable products incorporating atobacco matrix grown from culture. Such products, for example smokingarticles and smokeless products, optionally include conventionally-growntobacco in addition to the tobacco matrix. Tobacco products includelit-end (i.e., smokeable) and smokeless products. Lit-end productsinclude cigarettes, cigars, and the like, as well as loose tobaccointended for smoking (for example in a pipe or hand-rolled cigarette).

Smokeless tobacco products incorporating a tobacco matrix as disclosedherein may be in any format suitable for comfort in a consumer's oralcavity. Smokeless tobacco products contain tobacco in any form, such asmoist smokeless tobacco (MST), chewing tobacco, or orally-usable pouchedsmokeless tobacco products. The tobacco itself may take the form ofdried particles, shreds, granules, powders, or a slurry (i.e., tobaccoextract), deposited on, mixed in, surrounded by, or otherwise combinedwith other ingredients in any format, such as flakes, films, tabs,foams, or beads. Smokeless tobacco products may be wrapped with amaterial, which may be edible (i.e., orally disintegrable) ornon-edible. Liquid contents of smokeless tobacco products can beenclosed in a form, such as beads, to preclude interaction with awater-soluble wrapper. The wrapper may be shaped as a pouch to partiallyor completely enclose tobacco-incorporating compositions, and/or tofunction as an adhesive to hold together a plurality of tabs, beads, orflakes of tobacco, thereby forming a pouched tobacco product. A wrappermay also enclose a moldable tobacco composition that conforms to theshape of a consumer's mouth. An orally disintegrable wrapper may enclosesmokeless tobacco and may be formed on continuous thermoforming orhorizontal form/fill/seal equipment or other suitable packagingequipment using edible films (which may or may not contain tobacco).Exemplary materials for constructing a wrapper include film compositionscomprising hydroxypropyl methylcellulose (“HPMC”),carboxymethylcellulose (“CMC”), pectin, alginates, pullulan, and othercommercially viable, edible film-forming polymers. Other wrappingmaterials may include pre-formed capsules produced from gelatin, HPMC,starch/carrageenan, or other commercially available materials. Suchwrapping materials may include tobacco as an ingredient. Wrappers thatare not orally disintegrable may be composed of woven or nonwovenfabrics, of coated or uncoated paper, or of perforated or otherwiseporous plastic films. Wrappers may incorporate flavoring and/or coloringagents. Smokeless products can be assembled together with a wrapperutilizing any method known to persons skilled in the art of commercialpackaging, including methods such as blister packing and stick-packing,in which a small package can be formed by a vertical form/fill/sealpackaging machine.

It may also be advantageous to add one or more additional components orother additives to a tobacco product as described herein. Thesecomponents may include, but are not limited to, the following:flavorants, colorants, sweeteners, such as xylitol, bulking agents,fillers, anti-adherent compounds, dispersing agents, moisture absorbingcompounds, binder, chemesthetic agents, such as warming agents orcooling agents, and film-forming agents. Such components may be includedin any or all portions of the product, for example in a tobacco fillerand/or a wrapper. Such components may be used in amounts sufficient toachieve their intended effects.

Products including a tobacco matrix as described herein may also includea blend of conventionally-grown tobacco, non-tobacco smoking material,tobacco extender, tobacco dust, and/or reconstituted tobacco.

EXAMPLES Example 1 Initiation of Tobacco Callus Cultures from Explants

Preparation of Growth Media: Tobacco cultures can be stably establishedand propagated indefinitely in many types of media, including Murashigeand Skoog (MS), a medium commonly utilized for providing nutrients tovarious plant-tissue cultures. MS is regarded as a ‘high salt’ medium,and can be obtained from various commercial sources as a powder or aliquid concentrate. Growth regulators can be purchased similarly fromvarious commercial vendors. Methods for accurate and reproduciblepreparation of growth medium are known to persons skilled in the art.Reagents obtainable from reliable vendors include: Callus Media agarplates (100×20 mm) from Teknova No:2M9280; Murashige and Skoog BasalMedia with 2% Sucrose from Teknova No:(1 L); 2,4-D(2,4-Dichlorophenoxyacetic Acid from Sigma, Cat. No: D-6679 (25 g); IAA(Indole-3-acetic acid, from Sigma, Cat. No: 12886. MS Basal LiquidMedium with 2% Sucrose+1.5 μg/ml IAA can be prepared by combining 1.0 mlof a 1.5 mg/ml IAA solution into 1 L of MS Basal Media with 2% sucrose,under aseptic conditions. The media should be mixed well, and allowed toequilibrate for approximately 10-15 minutes prior to contact with cells.Toshio Murashige and Folke Skoog, “A Revised Medium for Rapid Growth andBio Assays with Tobacco Tissue Cultures,” Physiologia Plantarum, Vol.15, pp. 473-497 (1962), which is incorporated herein by reference in itsentirety.

Initiation of Explants: Under aseptic conditions, the terminal (e.g., 12inches) portion of the main stem of a tobacco plant can be severed,removing all leaves and side buds. The stem can be further divided into4 smaller pieces and placed in a beaker containing 70% ethanol (madewith sterile water) for 3 minutes. The stem pieces are placed in sterilewater three times, using sterile 1 L beakers for washes. A cylinder ofpith can be removed and placed into a sterile Petri dish using a sterilecork borer. About ¼ of an inch can be removed from each side of thecylinder of the pith. Using a sterile scalpel and forceps, the pith canbe further sliced into thin discs, approximately 1-2 mm thick. The discscan be placed, cut-side down, onto MS agar plates supplemented with 2,4D. Parafilm® can be wrapped around the lid of plates, and the platesincubated at 27° C. in the dark for approximately 1 month.

Sub-culturing of Callus Tissue: Sharp forceps or a sterile scalpel canbe used to divide callus tissue. Approximately 2 weeks after theexplants have formed a substantial callus, each piece can be dividedinto two or three pieces no less than 10 mm×10 mm. The pith can beremoved at this point. Using sterile forceps, the friable callus can beremoved from around the pith, and placed onto a new MS agar plate. Thiscan be repeated until all calluses from the old plate have beentransferred to new plates. Parafilm® can be wrapped around the lids ofnew plates and the plates incubated in the dark at 27° C.

Example 2 Initiation of Tobacco Cell Suspension Cultures from CallusTissue

Initiation of Suspension Cultures: The developing callus can betransferred to a 1 L sterile baffle bottom flask filled with 500 ml MSliquid media using sterilized forceps. Sufficient callus tissue can betransferred in small pieces to initiate a good suspension culture (i.e.,covering the top of liquid media). The flask can be capped lightly, andplaced in a dark shaking incubator set at 110-112 rpm at 27° C.

Sub-culturing of Suspension Cultures: The suspension can be transferredto new media, every 7-10 days, when the suspension culture becomes veryopaque. A new flask containing 300 ml of MS liquid media with 150 ml ofthe growing culture can be initiated by adding the liquid media to thesuspension culture in a 2-to-1 ratio. The growing suspension culture tobe transferred should be measured, for example, utilizing a 100 mldisposable graduated cylinder. The resulting culture should have ayellow, slightly thick appearance. To avoid anoxic or dying cells, thesuspension cultures should not be resting without shaking for more than30 min. MS liquid media can be added to original cultures for easierinoculation of new flasks, if necessary.

Example 3 Harvesting Tobacco Cells from Suspension Cultures

After at least 3 passages in liquid media, the flask containing tobaccocells can be harvested by various methods, including filtration, whichcan be extremely efficient. The entire content of the flask can bepoured through a filter unit (medium Buchner funnel with nylon meshfilter material) while applying a vacuum. The sample is washed in thefilter unit by adding 100 ml of tap water, and the remaining liquid canbe removed with the vacuum. This wash can be followed by two washes with100 ml of MilliQ® water. After the final wash, the sample can be allowedto air dry as much as possible. Subsequently, samples can befreeze-dried, or can be subjected to equivalent procedures. Preferably,the resulting material is stored under dry conditions to prevent uptakeof moisture.

Although the invention has been described with reference to particularembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. The various parts of the disclosure including the abstract,summary, and the title are not to be construed as limiting the scope ofthe present invention, as their purpose is to enable the appropriateauthorities, as well as the general public, to quickly determine thegeneral nature of the invention. Unless the term “means” is expresslyused, none of the features or elements recited herein should beconstrued as means-plus-function limitations pursuant to 35 U.S.C. §112,¶6. Accordingly, the invention is limited only by the claims.

What is claimed is:
 1. A method for producing a product, comprising thesteps of: growing tobacco cells in liquid suspension culture underconditions of osmotic stress and/or saline stress to form a culturematerial comprising tobacco cells and extracellular matrix; harvestingthe culture material; drying the culture material to create a tobaccomatrix; and incorporating the tobacco matrix into a product.
 2. Themethod of claim 1, wherein the tobacco matrix is substantially devoid ofdark pigment.
 3. The method of claim 1, wherein the growing is in theabsence of light and in the presence of a carbon source.
 4. The methodof claim 1, wherein the drying comprises freeze drying.
 5. The method ofclaim 1, wherein the tobacco matrix comprises at least 5% by weightextracellular matrix.
 6. The method of claim 1, wherein the liquidsuspension culture comprises at least 300 mM NaCl, at least 10% of apolymer of ethylene oxide, or both.
 7. The method of claim 1, comprisinga step of establishing a liquid suspension culture of tobacco cells froma callus culture.
 8. The method of claim 1, wherein the growing isperformed in the presence of a flavorant and/or flavorant precursor. 9.The method of claim 1, wherein the product is a lit-end tobacco productor a smokeless tobacco product.
 10. The method of claim 1, wherein thetobacco matrix has substantially reduced harsh, astringent, and/orbitter flavors as compared to conventionally-grown tobacco.
 11. Themethod of claim 1, wherein the product comprises: (a) a blend of saidmatrix and one or more components selected from the group consisting ofconventionally-grown tobacco, non-tobacco smoking material, tobaccoextender, tobacco dust, and reconstituted tobacco; (b) one or morecomponents selected from the group consisting of flavorants, colorants,sweeteners, bulking agents, fillers, anti-adherent compounds, dispersingagents, moisture absorbing compounds, binder, chemesthetic agents, andfilm-forming agents; or (c) both (a) and (b).
 12. A method for producinga product, comprising the steps of: growing tobacco cells in liquidsuspension culture in the absence of light and under conditions ofosmotic stress and/or saline stress to form a culture materialcomprising tobacco cells and extracellular matrix; harvesting theculture material; drying the culture material to create a tobacco matrixwherein the tobacco matrix is substantially devoid of dark pigment; andincorporating the tobacco matrix into a product
 13. A consumable productcomprising a matrix of cultured tobacco cells grown in vitro, whereinthe matrix of cultured tobacco cells comprises at least 5% by weightextracellular matrix.
 14. The product of claim 13, wherein the matrix ofcultured tobacco cells has substantially reduced harsh, astringent,and/or bitter flavors as compared to conventionally-grown tobacco. 15.The product of claim 13, wherein the product comprises a blend of saidmatrix and one or more components selected from the group consisting ofconventionally-grown tobacco, non-tobacco smoking material, tobaccoextender, tobacco dust, and reconstituted tobacco.
 16. The product ofclaim 13, wherein the cultured tobacco matrix is derived from a cultureof genetically-modified tobacco.
 17. The product of claim 13, whereinthe product further comprises one or more components selected from thegroup consisting of flavorants, colorants, sweeteners, bulking agents,fillers, anti-adherent compounds, dispersing agents, moisture absorbingcompounds, binder, chemesthetic agents, and film-forming agents.
 18. Theproduct of claim 13, wherein the product is a smokeless tobacco productor a lit-end tobacco product.
 19. The product of claim 17, wherein theproduct is an orally-usable pouched smokeless tobacco product.